Systems for the detecting pathogens II Flashcards
2 ways to detect genes or gene products that are pathogen specific?
Nucleic acid amplification techniques (NAAT)
Polymerase Chain Reaction (PCR)
PCR
-exponentially amplifying specific sections DNA via primers
-Two DNA primers (18-20bp) specific for opposite DNA strands, used to amplify
DNA region
-Product is visualised by fluorescent tags or staining in gels
qPCR?
Measures the speed at which a PCR amplicon product accumulates by the amount of fluorescence released
specificity and a good proximation of quantitation
Strand Displacement Amplification (SDA)
Using primers and producing a flourescent signal
-used for chlamydia and gonorrhoea routinely in the lab
what makes a gene a good target?
- sensitivity = you want a gene that is in more than 1 copy per organism
- specific = has to be specific primers that only work in that organism
- repetitive
what makes a test good?
Specificity
Is the test unique to the genus/species?
Accuracy
Do we need to detect live organisms?
Is the detection system susceptible to genomic shifts/mutations?
Rapidity
Is the result generated going to be beneficial to the patient?
Microarrays
Ordered short oligonucleotide probes (40-70mer) attached to slides in defined spots.
Each spot represents a single gene
Comparative Genomic Hybridisation (CGH) used mostly for DNA
Advantages
Covers the whole genome and strand dependant
Mass Spectrometry
-we don’t have to look for DNA, we could look at what the organism is consistent of, or something on the cell surface or something its producing that could be unique to that organism
- sample is broken up, ionised in flight and pushed against accelerator, affects its time of flight to the detector, produces peaks
- each peaks will be representative of one peptide (or something equivalent), makes a pattern
can compare the peaks to a database and work out if you’ve seen it before
- very fast and accurate
- will only identify known profiles: dependent on us knowing what we are looking for, otherwise we will get a pattern we’ve never seen before
- requires pure culture
- different organisms have different patterns
biomarkers?
body reacting to pathogens (eg. antibodies)
biomarkers can be against things in the cell wall or from the membrane, or peptidoglycan inside the membrane
eg. CSF Direct Agglutination test
- little beads have antibodies made against them
- agglutination happens depending on antibody specificity
- very fast, 30 seconds, simple
Serotyping and Toxin detection
biomarkers - toxin detection - how does this happen?
Enterohaemolysis
Agglutination with anti-toxin antibodies
PCR for the presence of the gene
adv and disadv of biomarkers?
Advantages
Good Specificity
Good Sensitivity
Easily automated
Disadvantages
Serological response is not rapid, therefore not useful in acute infections
Single sera results are meaningless due to possible previous exposure
Some antibodies are cross-reactive
Virulence is only INFERRED by the presence of a biomarker
ONLY in vivo testing of cultured pathogen
infected into an animal model can prove virulence
what can direct sequencing show?
differences between SINGLE bases in strains
Or resistance mutations to antibiotics
Bio-signature profiling?
Following the host transcriptomic profile with microarrays
-which one of your genes is turned on when you have that disease?
Metabolic profiling?
Also looking at how the body has reacted, not just looking at genes
- e.g. smell – training bees to smell who has tuberculosis
- TB patients exude a volatile fatty acid, and trained bees to smell this
- really cheap, quick, easy
name a future method
Rapid Point of Care testing
- Little chips (you can buy them in the chemist)
- take blood, urine
- it will take and lyse the sample, take out the DNA, it will PCR it and take a signal out of it and it will sequence the signal which puts it in your mobile phone and sends it to the lab, which will reply back with your results eg Yes, you have HIV
